Identification of Autographa californica nucleopolyhedrovirus ac93 as a core gene and its requirement for intranuclear microvesicle formation and nuclear egress of nucleocapsids

Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusion-derived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.